Plant thin cell layers (TCLs), which consist of a few layers of cells, are typically 0.5–1 mm thick. After the first application of TCLs to tobacco in 1943, and after 45 years of their practical application to plant tissue culture, TCLs have proven to be an effective tool for the in vitro culture of dozens of plant species. Their higher productivity compared to conventional explants has made TCLs continually relevant and useful in plant research and applied plant biotechnology. On the 45th anniversary of TCLs, this mini-review highlights the emergence of about a dozen new studies published in the past three years (2016–2018) that employed TCLs in either basic tissue culture or applied in vitro biotechnology. These new results emerged for medicinal plants (Bacopa monnieri, Scutellaria ocmulgee, Urginea altissima, Withania coagulans ), fruit trees (Rubus spp.), vegetables (Allium ascalonicum, Allium schoenoprasum), a conifer (Pinus patula ), ornamental plants including Lilium and three orchids (Dendrobium aqueum, Dendrobium aphyllum, Malaxis wallichii ), and a model plant (Arabidopsis thaliana ). In plant biotechnology, TCLs have a rich history and their continued use and future application to plant tissue culture or genetic transformation is promising.